MeCP2 plays a critical role in interpreting epigenetic signatures that command chromatin conformation and regulation of gene transcription. In spite of MeCP2's ubiquitous expression, its functions have always been considered in the context of brain physiology. In this study, we demonstrate that alterations of the normal pattern of expression of MeCP2 in cardiac and skeletal tissues are detrimental for normal development. Overexpression of MeCP2 in the mouse heart leads to embryonic lethality with cardiac septum hypertrophy and dysregulated expression of MeCP2 in skeletal tissue produces severe malformations. We further show that MeCP2's expression in the heart is developmentally regulated; further suggesting that it plays a key role in regulating transcriptional programs in non-neural tissues.
Chronic renal failure causes left ventricular hypertrophy, but the molecular mechanisms involved remain unknown. We, therefore, investigated whether the mineralocorticoid receptor is implicated in the cardiac hypertrophy observed in uremic rats and whether mineralocorticoid receptor blockade could be protective in chronic renal failure. Experimental groups were: control rats, uremic rats (NPX) with 5/6 nephrectomy (5 weeks), and NPX rats fed with spironolactone for 5 weeks. Systolic blood pressure was increased in both NPX rats and NPX rats fed with spironolactone for 5 weeks. Echocardiography revealed concentric left ventricular hypertrophy in uremia, which was attenuated by spironolactone. Enlarged cardiomyocyte size was observed in both left and right ventricles of NPX rats, an effect that was prevented by spironolactone. Mineralocorticoid receptor antagonism attenuated the increase of ventricular brain natriuretic peptide mRNA levels induced by nephrectomy. Left ventricular gene expressions of aldosterone synthase, mineralocorticoid receptor, and hydroxysteroid dehydrogenase type 2 were the same in the 3 groups, whereas gene expression of the glucocorticoid receptor was significantly diminished in chronic renal failure rats. No significant differences in cardiac aldosterone were observed between control rats and NPX rats, although NPX rats fed with spironolactone for 5 weeks showed increased plasma aldosterone levels. However, a significant increase in serum and glucocorticoid-inducible kinase-1 mRNA expression and protein was present in the NPX group; spironolactone treatment significantly reduced serum and glucocorticoid-inducible kinase-1 mRNA and protein in the left ventricle. Uremic rats exhibited a significant increase of superoxide production and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits expression (NOX-2, NOX-4, and p47 phox ) in the left ventricle, which was prevented by the mineralocorticoid receptor antagonist. Our findings provide evidence of the beneficial effects of spironolactone in cardiac hypertrophy and cardiac oxidative stress in chronic renal failure.
Adaptive immune response has been implicated in inflammation and fibrosis as a result of exposure to mineralocorticoids and a high-salt diet. We hypothesized that in mineralocorticoid-salt–induced hypertension, activation of the mineralocorticoid receptor alters the T-helper 17 lymphocyte (Th17)/regulatory T-lymphocyte/interleukin-17 (IL-17) pathway, contributing to cardiac and renal damage. We studied the inflammatory response and tissue damage in rats treated with deoxycorticosterone acetate and high-salt diet (DOCA–salt), with or without mineralocorticoid receptor inhibition by spironolactone. To determine whether Th17 differentiation in DOCA–salt rats is caused by hypertension per se, DOCA–salt rats received antihypertensive therapy. In addition, to evaluate the pathogenic role of IL-17 in hypertension and tissue damage, we studied the effect of IL-17 blockade with a specific antibody (anti–IL-17). We found activation of Th17 cells and downregulation of forkhead box P3 mRNA in peripheral tissues, heart, and kidneys of DOCA–salt–treated rats. Spironolactone treatment prevented Th17 cell activation and increased numbers of forkhead box P3–positive cells relative to DOCA–salt rats. Antihypertensive therapy did not ameliorate Th17 activation in rats. Treatment of DOCA–salt rats with anti–IL-17 significantly reduced arterial hypertension as well as expression of profibrotic and proinflammatory mediators and collagen deposits in the heart and kidney. We conclude that mineralocorticoid receptor activation alters the Th17/regulatory T-lymphocyte/IL-17 pathway in mineralocorticoid-dependent hypertension as part of an inflammatory mechanism contributing to fibrosis.
In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Ca2+i levels apparent both in the nucleus and cytosol, releasing this ion from intracellular stores through an inositol 1,4,5-trisphosphate (IP3)-dependent signaling pathway. Intracellular IP3 levels increased after IGF-1 stimulation in both the presence and absence of extracellular Ca2+. A different spatial distribution of IP3 receptor isoforms in cardiomyocytes was found. Ryanodine did not prevent the IGF-1-induced increase of Ca2+i levels but inhibited the basal and spontaneous Ca2+i oscillations observed when cardiac myocytes were incubated in Ca2+-containing resting media. Spatial analysis of fluorescence images of IGF-1-stimulated cardiomyocytes incubated in Ca2+-containing resting media showed an early increase in Ca2+i, initially localized in the nucleus. Calcium imaging suggested that part of the Ca2+ released by stimulation with IGF-1 was initially contained in the perinuclear region. The IGF-1-induced increase on Ca2+i levels was prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM, thapsigargin, xestospongin C, 2-aminoethoxy diphenyl borate, U-73122, pertussis toxin, and βARKct (a peptide inhibitor of Gβγ signaling). Pertussis toxin also prevented the IGF-1-dependent IP3 mass increase. Genistein treatment largely decreased the IGF-1-induced changes in both Ca2+i and IP3. LY29402 (but not PD98059) also prevented the IGF-1-dependent Ca2+i increase. Both pertussis toxin and U73122 prevented the IGF-1-dependent induction of both ERKs and protein kinase B. We conclude that IGF-1 increases Ca2+i levels in cultured cardiac myocytes through a Gβγ subunit of a pertussis toxin-sensitive G protein-PI3K-phospholipase C signaling pathway that involves participation of IP3. In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Ca2+i levels apparent both in the nucleus and cytosol, releasing this ion from intracellular stores through an inositol 1,4,5-trisphosphate (IP3)-dependent signaling pathway. Intracellular IP3 levels increased after IGF-1 stimulation in both the presence and absence of extracellular Ca2+. A different spatial distribution of IP3 receptor isoforms in cardiomyocytes was found. Ryanodine did not prevent the IGF-1-induced increase of Ca2+i levels but inhibited the basal and spontaneous Ca2+i oscillations observed when cardiac myocytes were incubated in Ca2+-containing resting media. Spatial analysis of fluorescence images of IGF-1-stimulated cardiomyocytes incubated in Ca2+-containing resting media showed an early increase in Ca2+i, initially localized in the nucleus. Calcium imaging suggested that part of the Ca2+ released by stimulation with IGF-1 was initially contained in the perinuclear region. The IGF-1-induced increase on Ca2+i levels was prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM, thapsigargin, xestospongin C, 2-aminoethoxy diphenyl borate, U-73122, pertussis toxin, and βARKct (a peptide inhibitor of Gβγ signaling). Pertussis toxin also prevented the IGF-1-dependent IP3 mass increase. Genistein treatment largely decreased the IGF-1-induced changes in both Ca2+i and IP3. LY29402 (but not PD98059) also prevented the IGF-1-dependent Ca2+i increase. Both pertussis toxin and U73122 prevented the IGF-1-dependent induction of both ERKs and protein kinase B. We conclude that IGF-1 increases Ca2+i levels in cultured cardiac myocytes through a Gβγ subunit of a pertussis toxin-sensitive G protein-PI3K-phospholipase C signaling pathway that involves participation of IP3. Insulin-like growth factor-1 (IGF-1) 1The abbreviations used are: IGF-1, insulin-like growth factor 1; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; PKB, protein kinase B; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; PTX, pertussis toxin; MES, 2-(N-morpholino)ethanesulfonic acid; Ad adenovirus; [Ca2+]i, intracellular [Ca2+]; TBST, Tris-buffered saline with Tween 20; ROI, region of interest; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. plays important roles in numerous physiological processes, ranging from normal growth and development during the early stages of embryogenesis to the regulation of specific functions in several tissues and organs in later stages of development (1Liu J.L. Yakar S. LeRoith D. Endocrinology. 2000; 141: 4436-4441Crossref PubMed Scopus (54) Google Scholar). 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Following IGF-1 binding, IGF-1R becomes autophosphorylated on several tyrosine residues, allowing its interaction with phosphotyrosine binding or Src homology 2 (SH2) domain-containing proteins, thereby transducing signals to downstream effectors (11Dupont J. LeRoith D. Horm. Res. 2001; 55: 22-26Crossref PubMed Scopus (175) Google Scholar). Through these effectors, IGF-1 activates two main signaling cascades, the Ras-Rafmitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathways (12Petley T. Graff K. Jiang W. Yang H. Florini J. Horm. Metab. Res. 1999; 31: 70-76Crossref PubMed Scopus (104) Google Scholar). Several recent studies have demonstrated that IGF-1R also activates heterotrimeric G proteins in some cell types. Luttrell et al. (13Luttrell L.M. van Biesen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) first demonstrated in rat-1 fibroblasts that treatment with pertussis toxin (PTX) or transfection with a Gβγ scavenger (βARK-CT) blocked IGF-1-induced ERK activation (13Luttrell L.M. van Biesen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). This study suggested the involvement of the Gβγ subunit of a PTX-sensitive G protein in the activation of ERK (13Luttrell L.M. van Biesen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Similar results have been obtained with human intestinal smooth muscle cells (14Kuemmerle J.F. Murthy K.S. J. Biol. Chem. 2001; 276: 7187-7194Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), 3T3-L1 mouse pre-adipose cells (15Dalle S. Ricketts W. Imamura T. Vollenweider P. Olefsky J.M. J. Biol. Chem. 2001; 276: 15688-15695Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and rat cerebellar granule neurons (16Hallak H. Seiler A.E. Green J.S. Ross B.N. Rubin R. J. Biol. Chem. 2000; 275: 2255-2258Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Conflicting observations on the roles of intracellular calcium (Ca2+) and inositol 1,4,5-trisphosphate (IP3) in IGF-1 signaling have been reported. In BALB/c 3T3 cells, IGF-1 stimulates Ca2+ influx via IGF-1R by activating a Ca2+-permeable cation channel (17Kojima I. Matsunaga H. Kurokawa K. Ogata E. Nishimoto I. J. Biol. Chem. 1988; 263: 16561-16567Abstract Full Text PDF PubMed Google Scholar). The resulting Ca2+ influx produces oscillations in the concentration of free intracellular Ca2+ ([Ca2+]i) independently of phosphoinositide turnover (18Kojima I. Mogami H. Ogata E. Am. J. Physiol. 1992; 262: E307-E311PubMed Google Scholar). In contrast, in cultured bovine alveolar macrophages, nanomolar concentrations of IGF-1 stimulate after 30 s the accumulation of IP3 and inositol 1,3,4,5-tetraphosphate, and induce a rise in [Ca2+]i (19Geertz R. Kiess W. Kessler U. Hoeflich A. Tarnok A. Gercken G. Mol. Cell. Biochem. 1997; 177: 33-45Crossref PubMed Google Scholar). In chondrocytes, IGF-1 induces Ca2+ release from the endoplasmic reticulum, which is partially blocked by phospholipase C (PLC) inhibitors and PTX (20Poiraudeau S. Lieberherr M. Kergosie N. Corvol M.T. J. Cell. Biochem. 1997; 64: 414-422Crossref PubMed Scopus (51) Google Scholar). Short term exposure of neurons and neuronal cell lines to IGF-1 leads to direct activation of voltage-gated L-type Ca2+ channels (21Selinfreund R.H. Blair L.A. Mol. Pharmacol. 1994; 45: 1215-1220PubMed Google Scholar, 22Blair L.A. Marshall J. Neuron. 1997; 19: 421-429Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 23Kleppisch T. Klinz F.J. Hescheler J. Brain Res. 1992; 591: 283-288Crossref PubMed Scopus (30) Google Scholar), whereas in rat pinealocytes IGF-1 inhibits L-type currents (24Chik C.L. Li B. Karpinski E. Ho A.K. Endocrinology. 1997; 138: 2033-2042Crossref PubMed Scopus (30) Google Scholar). IGF-1 also modulates L-type Ca2+ channels in adult rat cardiac myocytes and in skeletal muscle of young and middle-aged rats, but not of aged rats (25Renganathan M. Sonntag W.E. Delbono O. Biochem. Biophys. Res. Commun. 1997; 235: 784-789Crossref PubMed Scopus (36) Google Scholar, 26Solem M.L. Thomas A.P. Biochem. Biophys. Res. Commun. 1998; 252: 151-155Crossref PubMed Scopus (27) Google Scholar). In whole heart and in ferret papillary muscle, IGF-1 displays an acute positive inotropic effect, which is secondary to augmented myofilament responsiveness to Ca2+ (27Cittadini A. Ishiguro Y. Strömer H. Spindler M. Moses A.C. Clark R. Douglas P.S. Ingwall J.S. Morgan J.P. Circ. Res. 1998; 83: 50-59Crossref PubMed Scopus (140) Google Scholar). In rat ventricular papillary muscle cells, however, acute exposure to IGF-1 or insulin results in enhanced muscle contractility that is associated with [Ca2+]i transients (28Freestone N.S. Ribaric S. Mason W.T. Mol. Cell. Biochem. 1996; 163–164: 223-229Crossref PubMed Scopus (102) Google Scholar). In cardiac myocytes, IGF-1 activates multiple signaling pathways, including ERK-PKC, PKB/PI3K, PLC-γ, and JAK-STAT (9Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (738) Google Scholar, 29Foncea R. Andersson M. Ketterman A. Blakesley V. Sapag-Hagar M. Sugden P.H. LeRoith D. Lavandero S. J. Biol. Chem. 1997; 272: 19115-19124Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 30Foncea R. Gálvez A. Pérez V. Morales M.P. Calixto A. Meléndez J. González-Jara F. Díaz-Araya G. Sapag-Hagar M. Sugden P.H. Le-Roith D. Lavandero S. Biochem. Biophys. Res. Commun. 2000; 273: 736-744Crossref PubMed Scopus (35) Google Scholar, 32Takahashi T. Fukuda K. Pan J. Kodama H. Sano M. Makino S. Kato T. Manabe T. Ogawa S. Circ. Res. 1999; 85: 884-891Crossref PubMed Scopus (48) Google Scholar). However, the role of cytosolic Ca2+ as the second messenger in the complex IGF-1 signaling pathways present in cardiac myocytes has not been examined. In the present study we show that, both in the presence or absence of extracellular Ca2+, addition of IGF-1 to cultured cardiac myocytes induced a rapid [Ca2+] increase in both the nucleus and cytoplasm and also produced a rapid increase of IP3 levels. The presence of extracellular Ca2+ modified the kinetics of the [Ca2+] increase, delaying an increase of cytosolic but not nuclear [Ca2+]. The role of tyrosine kinase, G-protein, PI3K, PLC, and the IP3 receptor in the pathway leading to these calcium signals was established using specific inhibitors. Materials—[3H]IP3 was from PerkinElmer Life Sciences. Fluo3-acetoxymethyl ester (Fluo3-AM) was from Molecular Probes (Eugene, OR). Thapsigargin, xestospongin C, genistein, BAPTA-acetoxymethyl ester (BAPTA-AM), LY294002 (LY), PD98059 (PD), and SB203580 (SB) were from Calbiochem-Novabiochem Corp. (San Diego, CA). 2-Aminoethoxy diphenyl borate was from Aldrich. Polyclonal antibodies against type 1 and type 2 IP3 receptor were from Affinity BioReagents (Golden, CO) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Antitype 3 IP3 receptor was kindly donated by G. A. Mignery (Loyola University, Chicago, IL). Polyclonal antibodies against phosphorylated (Ser473) PKB, total PKB, phosphorylated (Thr202/Thr204) ERK, and total ERK were from Cell Signaling Technology Inc. (Beverly, MA). Dulbecco's modified Eagle's medium, medium 199, U-73122, ryanodine, nifedipine, PTX, IP3, and other biochemicals were purchased from Sigma unless stated otherwise. Human recombinant IGF-1 was donated by Dr. C. George-Nascimento (Austral Biologicals, San Ramon, CA). Animals—Rats were bred in the Animal Breeding Facility from the Faculty of Chemical and Pharmaceutical Sciences, University of Chile (Santiago, Chile). We performed all studies with the approval of the institutional bioethical committee at the Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago. This investigation conforms to the "Guide for the Care and Use of Laboratory Animals" published by the United States National Institutes of Health (31United States National Institutes of HealthGuide for the Care and Use of Laboratory Animals. National Institutes of Health Publication 85-23, 1985Google Scholar). Culture of Cardiac Myocytes—Cardiac myocytes were prepared from hearts of 1–3-day-old Sprague-Dawley rats as described previously (29Foncea R. Andersson M. Ketterman A. Blakesley V. Sapag-Hagar M. Sugden P.H. LeRoith D. Lavandero S. J. Biol. Chem. 1997; 272: 19115-19124Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). For IP3 determination, cardiomyocytes were plated at a final density of 0.7 × 103/mm2 on gelatin-precoated 60-mm dishes. For detection of Ca2+, cells were plated with a final density of 1.0 × 103/mm2 on gelatin-precoated coverslips. Serum was withdrawn for 24 h before the cells were treated further with agonist IGF-1 (1–100 nm) in serum-free medium (Dulbecco's modified Eagle's medium/medium 199) at 37 °C. Cultured cardiomyocytes, assessed with an anti-β-myosin heavy chain antibody, were at least 95% pure. Recombinant Adenoviruses—Adenoviral vectors (Ad) were propagated and purified as previously described (33Shah A.S. White D.C. Emani S. Kypson A.P. Lilly R.E. Wilson K. Glower D.D. Lefkowitz R.J. Koch W.J. Circulation. 2001; 103: 1311-1316Crossref PubMed Scopus (175) Google Scholar). Two transgenes (a gift from Dr. W. J. Koch, Duke University Medical Center, Durham, NC) were used: βARKct (Ad-βARKct) and an empty viral construct (Ad-EV). βARKct is a peptide inhibitor of Gβγ signaling (34Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). Cardiomyocytes were infected with adenoviral vectors at a multiplicity of infection of 300. Determination of IP3 Mass—Cardiomyocytes were rinsed and preincubated for 20 min at room temperature in 58 mm NaCl, 4.7 mm KCl, 3 mm CaCl2, 1.2 mm MgSO4, 0.5 mm EDTA, 60 mm LiCl, 10 mm glucose, and 20 mm Hepes, pH 7.4. Cells were stimulated by fast (1 s) replacement of this solution by a solution containing IGF-1. At the times indicated, the reaction was stopped by rapid aspiration of the stimulating solution, addition of 0.8 m ice-cold perchloric acid and freezing with liquid nitrogen. Samples were allowed to thaw and cell debris was spun down for protein determination. The supernatant was neutralized with a solution of 2 m KOH, 0.1 m MES, and 15 mm EDTA. The neutralized extracts were frozen at –80 °C until required for IP3 determination. Measurements of IP3 mass were made by radioreceptor assay (35Liberona J.L. Powell J.A. Shenoi S. Petherbridge L. Caviedes R. Jaimovich E. Muscle Nerve. 1998; 21: 902-909Crossref PubMed Scopus (54) Google Scholar). Briefly, a crude rat cerebellum membrane preparation was obtained after homogenization of tissue in 50 mm Tris-HCl, pH 7.7, containing 1 mm EDTA, 2 mm β-mercaptoethanol and centrifugation at 20,000 × g for 15 min. This procedure was repeated 3 times, suspending the final pellet in the same solution plus 0.3 m sucrose and freezing it at –80 °C until required for use. The rat cerebellar membrane preparation was calibrated for IP3 binding with 1.6 nm [3H]IP3 and 2–120 nm cold IP3, with sample analysis performed in a similar way but replacing cold IP3 with a portion of the neutralized supernatant. [3H]IP3 radioactivity, which remained bound to membranes, was measured in a Beckman LS-6000TA liquid scintillation spectrometer (Beckman Instruments Corp., Fullerton, CA). Protein was determined by the Lowry method (36Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Measurement of Intracellular Calcium—Cellular calcium images were obtained from neonatal cardiac myocytes preloaded with Fluo3-AM, using an inverted confocal microscope (Carl Zeiss Axiovert 135 M-LSM Microsystems) or a fluorescence microscope (Olympus Diaphot-TMD, Nikon Corporation) equipped with a cooled CCD camera and image acquisition system (Spectra Source MCD 600). Cardiac myocytes were washed three times with Ca2+-containing resting media (Krebs buffer: 145 mm NaCl, 5 mm KCl, 2.6 mm CaCl2, 1 mm MgCl2, 10 mm HEPES-Na, 5.6 mm glucose, pH 7.4) to remove Dulbecco's modified Eagle's medium/medium 199 culture medium, and loaded with 5.4 μm Fluo3-AM (coming from a stock in 20% pluronic acid, Me2SO) for 30 min at room temperature. After loading, cardiac myocytes were washed either with the same buffer or with a Ca2+-free resting media (145 mm NaCl, 5 mm KCl, 1.0 mm EGTA, 1 mm MgCl2,10mm HEPES-Na, 5.6 mm glucose, pH 7.4) and used within 2 h. The cell-containing coverslips were mounted in a 1-ml capacity plastic chamber and placed in the microscope for fluorescence measurements after excitation with a 488-nm wavelength argon laser beam or filter system. IGF-1 was either added directly or the solution was fast (1 s) changed in the chamber. The fluorescent images were collected every 0.4–2.0 s for fast signals and analyzed frame by frame with the image data acquisition program (Spectra-Source) of the equipment. An objective lens PlanApo 60X (numerical aperture 1.4) was generally used. In most of the acquisitions, the image dimension was 512 × 120 pixels. Intracellular calcium was expressed as a percentage of fluorescence intensity relative to basal fluorescence (a value stable for at least 5 min in resting conditions). The fluorescence intensity increase is proportional to the rise in [Ca2+]i (37Minta A. Kao J.P. Tsien R.Y. J. Biol. Chem. 1989; 264: 8171-8178Abstract Full Text PDF PubMed Google Scholar). Digital Image Processing—Elimination of out-of-focus fluorescence was performed using both the "no neighbors" deconvolution algorithm and Castleman's (38Castleman K. Digital Image Processing. Prentice-Hall, Englewood Cliffs, NJ1989Google Scholar) point spread function theoretical model, as described previously (39Estrada M. Liberona J.L. Miranda M. Jaimovich E. Am. J. Physiol. 2000; 279: E132-E139Crossref PubMed Google Scholar). For quantitation of fluorescence, the summed pixel intensity was calculated from the section delimited by a contour. As a way of increasing efficiency of these data manipulations, action sequences were generated. To avoid interference in the fluorescence by possible IGF-1 effects on the cellular volume, the area of each fluorescent cell was determined by image analysis using adaptive contour and then creating a binary mask, which was compared with its bright-field image. Western Blot Analysis—Cell lysates were matched for proteins (10–40 μg) and were separated by SDS-PAGE on 10% polyacrylamide gels and electrotransferred to nitrocellulose. Membranes were blocked with 5% nonfat milk powder in Tris-buffered saline (TBS) (pH 7.6) containing 0.1% (v/v) Tween 20 (TBST) for 60 min at room temperature. Primary antibodies were diluted 1/1,000 in blocking solution. Nitrocellulose membranes were incubated with primary antibodies overnight at 4 °C. After washing in TBST (3× 10 min each), the blots were incubated for 2 h at room temperature with horseradish peroxidase-linked secondary antibody (1:5,000 in 1% (w/v) nonfat milk powder in TBST). The blots were washed again in TBST and the bands were detected using ECL with exposure to Kodak film for 0.5–30 min. Blots were quantified by scanning densitometry. Subcellular Fractionation—Nuclear and cytosolic fractions from cultured cardiac myocytes were prepared as described by Schreiber et al. (40Schreiber E. Matthias P. Müller M. Schaffer W. Nucleic Acids Res. 1989; 17: 6413-6422Crossref PubMed Scopus (3918) Google Scholar). Homogeneity of fractions were assessed by Western blot using α-actinin (marker for myofibril Z bands) (41Hilenski L.L. Terracio L. Borg T.K. Cell Tissue Res. 1991; 264: 577-587Crossref PubMed Scopus (63) Google Scholar), lamina-associated polypeptide 2 (marker for nuclear inner membrane) (42Vlcek S. Dechat T. Foisner R. Cell. Mol. Life Sci. 2001; 58: 1758-1765Crossref PubMed Scopus (58) Google Scholar), and calsequestrin (marker for sarcoplasmic reticulum) (43Yano K. Zarain-Herzberg A. Mol. Cell. Biochem. 1994; 135: 61-70Crossref PubMed Scopus (140) Google Scholar). Immunocytochemistry—Cardiomyocytes grown on coverslips were fixed in iced methanol, blocked in phosphate-buffered saline containing 1% bovine serum albumin for 30 min, and incubated with primary anti-IP3 receptor antibodies at 4 °C overnight. The cells were then washed 5-fold with phosphate-buffered saline/bovine serum albumin and incubated with the appropriate goat anti-rabbit secondary antibody for 1 h at room temperature. After washing three more times, the coverslips were mounted in Vectashield (Vector Laboratories, Inc.). The samples were evaluated in a scanning confocal microscope (Carl Zeiss Axiovert 135, LSM Microsystems) and documented through computerized images (44Estrada M. Cardenas C. Liberona J.L. Carrasco M.A. Mignery G.A. Allen P.D. Jaimovich E. J. Biol. Chem. 2001; 276: 22868-22874Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Expression of Results and Statistical Analysis—Data are represented as mean ± S.E. of the number of independent experiments indicated (n) or as examples of representative experiments performed on at least three separate occasions. Data were analyzed by analysis of variance and comparisons between groups were performed using a protected Tukey's t test. A value of p < 0.05 was set as the limit of statistical significance. IGF-1-induced Intracellular Ca2+Transients in Rat Cardiac Myocytes—Changes in intracellular Ca2+ concentration, [Ca2+]i, were visualized in single cardiomyocytes preloaded with Fluo3-AM. Increases of relative fluorescence represent an increase of free cytosolic [Ca2+]. Multiple region of interest (ROI) analysis of single cells revealed that similar kinetics and fluorescence intensities were detected disregarding the analyzed ROI window (data not shown). Quiescent cardiomyocytes, maintained in Ca2+-containing resting solution, exhibited basal [Ca2+]i oscillations that were blocked by 30 min preincubation with ryanodine (50 μm) (data not shown). Incubation of cardiomyocytes in resting solution without Ca2+ also prevented [Ca2+]i oscillations (data not shown). Addition of IGF-1 (1 nm) to cardiomyocytes maintained in Ca2+-free resting solution induced a fast and transient (10–20 s) increase in [Ca2+]i, with a maximum at 4–5 s after IGF-1 addition (Fig. 1, A and B). The lowest concentration of IGF-1 that induced [Ca2+]i transients was 0.01 nm. When cardiomyocytes were maintained in Ca2+-containing resting solution, addition of IGF-1 induced a similar, although slower increase of [Ca2+]i, reaching a maximum 40–50 s after IGF-1 addition (Fig. 1C). Cardiomyocytes maintained in Ca2+-containing resting solution usually showed basal [Ca2+]i oscillations; these oscillations were not altered by addition of IGF-1. Following IGF-1 addition in Ca2+-free resting solution, an increase of [Ca2+]i was observed both in the nucleus and cytoplasm (Fig. 1A). Analysis of ROI in the fluorescence images showed that both nuclear and cytosolic Ca2+ transient time courses were similar in IGF-1-stimulated cardiomyocytes maintained in the Ca2+-free resting solution (Fig. 1D). However, when cardiomyocytes were treated with IGF-1 in Ca2+-containing resting solution, nuclear [Ca2+] increased faster than cytosolic [Ca2+] (Fig. 1E). ROI analysis clearly showed that extracellular Ca2+ slowed down the cytosolic but not the nuclear increase in [Ca2+] stimulated by IGF-1 (Fig. 1, D and E). Rates of rise of the nuclear Ca2+ transients in the presence and absence of external Ca2+ were 32.3 ± 7.6 and 38.6 ± 8.1 (Δf/f per s) (n = 4), respectively. However, rates of rise of Ca2+ transients in the cytosol were 3.9 ± 1.4 and 20.1 ± 5.3 (Δf/f per s) (n = 10), respectively. In all cells studied, the IGF-1-induced increase in [Ca2+] did not trigger regenerative waves through the cytosol. IGF-1-induced Ca2+Release from Ryanodine and Nifedipine-insensitive Intracellular Stores—The increase in [Ca2+]i in cardiomyocytes upon stimulation with IGF-1 in the absence of extracellular Ca2+ suggests involvement of Ca2+ release from internal stores. Resting Fluo3-AM preloaded cardiomyocytes usually displayed increased basal fluorescence in the perinuclear region (Fig. 2, A and B). After addition of IGF-1, the distribution of fluorescence changed and the increase in fluorescence was homogeneous in both the nucleus and cytoplasm (Fig. 2B, 2 s and 10 s). These observations suggest that IGF-1 induced Ca2+ release from perinuclear Ca2+ stores. IGF-1-induced Ca2+ transients in cardiomyocytes maintained in Ca2+-free resting media were not suppressed by ryanodine (Fig. 3B). However, ROI analysis of nuclear and cytosolic fluorescence revealed that in the presence of ryanodine the increase in cytosolic [Ca2+] was slower than in the nucleus (Fig. 3D). Cardiomyocytes incubated in Ca2+-containing resting solution displayed an analogous behavior. These results suggest that the IGF-1 stimulated fast increase in cytoplasmic Ca2+ was partly because of calcium release through ryanodine receptors because a fast component was inhibited after preincubation with 20 μm ryanodine. Nifedipine did not modify IGF-1-induced Ca2+ transients in cardiomyocytes maintained in Ca2+-free resting solution (Fig. 3C). ROI analysis of images revealed that both nuclear and cytosolic fluorescence increases presented similar kinetics in the presence or absence of nifedipine (Fig. 3C). These results suggest that voltage-dependent L-type Ca2+ channels are not involved in the IGF-1-induced Ca2+ transients in cardiomyocytes. To further demonstrate the role of intracellular Ca2+ stores in the generation of IGF-1-induced Ca2+ transients, cardiomyocytes were preloaded with both BAPTA-AM and Fluo3-AM, and then stimulated with 1 nm IGF-1. BAPTA-AM inhibited the increase of fluorescence induced by IGF-1 and only a small decrease in fluorescence was apparent in these conditions (Fig. 4A). Treatment of Fluo3-AM-preloaded cardiomyocytes with thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, led to a slow increase of intracellular fluorescence because of Ca2+ loss from internal stores. Subsequent addition of IGF-1 (5 min) to these cells did not induce the characteristic increase in the fluorescence signal (Fig. 4B). Similar results were obtained when internal Ca2+ stores where depleted using caffeine, a ryanodine receptor activator (Fig. 4C). When thapsigargin-pretreated cardiomyocytes were incubated with caffeine, no additional Ca2+ release was detected (Fig. 4D). Further treatment with IGF-1 did not increase fluorescence, an effect obtained by the addition of external Ca2+ (Fig. 4D). This Ca2+ influx elicited by calcium add back suggests the activation of a plasma membrane store-operated channel activity in neonatal rat cardiac myocytes. However, external Ca2+ addition to cardiac myocytes after IGF-1 treatment showed a l
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